Reversible electrochemical sensors for polyions

ABSTRACT

The present invention is directed to a reversible electrochemical sensor for polyions. The sensor uses active extraction and ion stripping, which are controlled electrochemically. Spontaneous polyion extraction is suppressed by using membranes containing highly lipophilic electrolytes that possess no ion-exchange properties. Reversible extraction of polyions is induced by constant current pulse of fixed duration applied across the membrane. Subsequently, polyions are removed by applying a constant stripping potential. The sensors provide excellent stability and reversibility and allow for measurements of heparin concentration in whole blood samples via protamine titration. The sensors can also monitor a polyion concentration and an enzyme activity, wherein the polyion decomposition is directly proportional to the enzyme activity in a sample. Additionally, the sensors can monitor an enzyme inhibitor activity. Also, an immunoassay can be used to detect analytes by employing one of a polyion and an enzyme as markers.

RELATED APPLICATIONS

This patent application is a continuation-in-part application of U.S.patent application Ser. No. 10/887,251, filed on Jul. 8, 2004 andentitled REVERSIBLE ELECTROCHEMICAL SENSORS FOR POLYIONS, which claimsbenefit of priority of U.S. Provisional Application No. 60/485,856,filed on Jul. 9, 2003. U.S. patent application Ser. No. 10/887,251,filed on Jul. 8, 2004 and U.S. Provisional Application No. 60/485,856,filed on Jul. 9, 2003, are both herein incorporated by reference intheir entirety.

This patent application also claims priority under 35 U.S.C. §119(e) ofthe co-pending U.S. Provisional Patent Application Ser. No. 60/706,117,filed on Aug. 5, 2005, and titled ELECTROCHEMICAL DETECTION OF ENZYMEACTIVITIES USING POLYIONS AS SUBSTRATE AND POLYION-SELECTIVE REVERSIBLEELECTROCHEMICAL SENSORS, which is incorporated herein by reference inits entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The research underlying this invention was supported in part with fundsfrom National Institutes of Health (NIH) grant nos. GM071623 andEB002189. The United States Government may have an interest in thesubject matter of this invention.

FIELD OF THE INVENTION

The present invention is directed to polyion sensors. The invention isfurther directed to membranes for use in the detection of polyions, suchas protamine and heparin, and to methods of such detection throughincorporation of the membranes in electrochemical cells. In particular,the invention relates to detection of polyions through forced movementof the polyions across the membrane, wherein the movement of thepolyions is reversible allowing for reuse of the membrane.

BACKGROUND

In the past decade a new direction in the field of ion-selectiveelectrodes has emerged with the development of potentiometric sensorswith plasticized polymeric membranes for the detection of polyionicmacromolecules. Early work in this area proposed a polymer membraneelectrode containing a lipophilic anion-exchanger, which was capable ofdetecting the polyanion heparin. See Ma, S. C., Yang, V. C., andMeyerhoff, M. E. Anal. Chem. 1992, 64, 694. Heparin-selective polymericmembrane electrodes are further described in U.S. Pat. No. 5,236,570 andU.S. Pat. No. 5,453,171.

Heparin is a highly sulfated polysaccharide with an average charge of−70 and an average molecular weight of 15,000 Daltons. The molecularformula for one unit of a heparin compound is provided below.

Heparin is used as an anticoagulant in major surgical and extracorporealprocedures, such as open-heart surgery, bypass surgery, and dialysis.The use of excess heparin in medical procedures can be detrimental,however, necessitating precise monitoring of heparin administration.Real-time monitoring of heparin concentration in blood is particularlyuseful for preventing the risk of excessive bleeding during operationsand reducing postoperative complications. Activated clotting timemeasurement (ACT) is a common method for estimating the heparinconcentration in whole blood. Although this method is widely used, it isnonspecific and indirect, and the results can be affected by manyvariables. In contrast to ACT, the heparin-selective electrode is ableto detect heparin concentration directly in whole blood or plasmasamples.

Similarly, an electrode for sensing the polycation protamine has alsobeen proposed. See Yun, J. H., Meyerhoff, M. E., and Yang, V. C. AnalBiochem. 1995, 224, 212. The polypeptide protamine is generally used forneutralization of heparin activity (i.e. to promote coagulation).Protamine, which is illustrated below, is a polycation with an averagecharge +20 and is rich in arginine residues.

The basic guanidinium groups of protamine complex electrostatically withthe sulfonate groups of heparin to render the anticoagulant activity ofthe heparin ineffective. Excess use of protamine, however, can also bedetrimental. For example, the use of protamine frequently results inadverse hemodynamic and hematologic side effects, such as hypertension,depressed oxygen consumption, thrombocytopenia with pulmonary plateletsequestration, and leukopenia. It is therefore useful to be able toaccurately detect and measure protamine concentration in biologicalfluid, such as blood.

Reliable detection of protamine allows for careful administration of theagent, thereby avoiding the associated problems noted above. Further,with the ability to detect protamine via ion-selective electrodes, it isalso possible to determine the heparin concentration in a sample viatitration of the sample with protamine. This is possible due to thespecific heparin-protamine interactions described above. Such action isalso described by Ramamurthy, et al., Clin. Chem. 1998, 606.

The observed response of the heparin-specific membrane electrode knownin the art could not be explained in terms of classic equilibriumapproach. The Nernst equation should yield a slope of the electrodefunction of less than 1 mV/decade and 2 mV/decade for heparin andprotamine respectively, because of the high charge of these ions. Aquasi-steady-state model to explain this unusual mechanism wassubsequently described. See Fu, B. et al., Anal Chem. 1994, 66, 2250.The potentiometric polyion sensor response is kinetic in nature. Astrong flux of polyions occurs both in the aqueous solution and themembrane phase due to the spontaneous extraction of polyions into thepolymeric membrane and the concomitant exchange with hydrophilic ionsfrom the membrane, which results in a potential change in the presenceof polyions.

Because the extraction of polyions is an irreversible process when usingthe heparin-specific membrane electrode of the prior art, a strongpotential drift is normally observed. After a relatively short time incontact with a polyion solution the sensor starts to lose its response.Extracted polyions must be removed from the membrane phase byreconditioning of the sensor, such as in concentrated sodium chloridesolution. Multiple methods have been proposed in the art for overcomingresponse loss due to polyion concentration at the membrane surface. A pHcross-sensitive potentiometric heparin sensor has been proposed, whereinthe sensor contains an ion-exchanger and a charged H+ ionophore.According to this method, heparin stripping could be accomplished byadjusting the pH of the sample. Another approach for overcoming lostsensor response is to use disposable sensors.

Thus, despite the existence of a selective extraction principle, it hasbeen impossible thus far to design a reversible polyion sensor.Accordingly, while polyion sensors can be highly useful in critical careapplications, their use is limited by the quick loss of response of thesensor. Single use sensors lead to increased expense, and the necessityof removing the sensor and reconditioning the sensor by a separatemethods is overly time consuming and adversely limiting on theusefulness of the sensor. Therefore, it would be useful to have a sensorfor detecting polyions that is fully reversible, wherein such reversalcan be performed quickly, repeatedly, and without removing the sensor toa separate solution.

SUMMARY OF THE INVENTION

The present invention provides reversible electrochemical sensors forpolyions. The sensors incorporate a potentiometric response mechanismfor determining the concentration of the polyion analyte, but theprocesses of extraction and ion stripping are controlledelectrochemically. Spontaneous polyion extraction is suppressed by usingmembranes containing highly lipophilic electrolytes that possess noion-exchange properties. Reversible extraction of polyions is induced ifa constant current pulse of fixed duration is applied across themembrane electrode of the invention. Subsequently, polyions are removedby applying a constant stripping potential. This ability to strip thepolyions, effectively regenerating the sensor, solves the problem facedby previously proposed polyion sensors that were prone to drifting andrequired prolonged contact with concentrated salt solutions to strip thepolyions from the sensing membrane before another measurement could betaken.

Membranes comprising the lipophilic electrolytes can be used withelectrochemical cells for continuous measurement of polyionconcentration in a sample solution without removing the electrode forreconditioning or replacing the electrode. Accordingly, titrations ofpolyions, such as heparin, are possible. For example, determination ofheparin concentration in whole blood samples is possible using protaminetitration. According to one aspect of the present invention, there isprovided a polyion-selective membrane for use in an electrochemicalcell, wherein the membrane comprises a lipophilic electrolyte having alipophilic cation component and a lipophilic anion component.Preferentially, one of the lipophilic cation component and thelipophilic anion component is selective for a specific polyion. Polyionsthat are particularly desirable for detection according to the presentinvention are heparin and protamine. Additional polyions that may bedetected with the membrane of the present invention includedeoxyribonucleic acids (DNA), ribonucleic acids (RNA), humic acids,carrageenans, and other polyionic macromolecules.

As noted above, one of the lipophilic cation component and thelipophilic anion component of the lipophilic electrolyte is selectivefor a particular polyion. Accordingly, in one embodiment of theinvention, the lipophilic electrolyte comprises a lipophilic anioncomponent that is selective for protamine. Preferentially, in thisembodiment, the lipophilic electrolyte is tetradodecylammonium1,3-dinonylnaphthalene-4-s-ulfonate (TDDA-DNNS). Similarly, in anotherembodiment, the lipophilic electrolyte comprises a lipophilic cationcomponent that is selective for heparin. Preferentially, in thisembodiment, the lipophilic electrolyte is dodecylguanidiniumtetrakis(p-chlorophenyl)borate (DDG-TCIPB).

In another embodiment of the invention, there is provided apolyion-selective membrane comprising a polymeric film-forming material,a plasticizer, and a lipophilic electrolyte having a lipophilic cationcomponent and a lipophilic anion component, wherein one of thelipophilic cation and lipophilic anion components is selective for aspecific polyion. Preferentially, the polymeric film-forming material ispolyvinyl chloride and the plasticizer is 2-nitrophenyl octyl ether.

In still another embodiment according to the present invention, apolyion-selective membrane for use in an electrochemical cell isprovided, wherein the membrane comprises a microporous hydrophobicsubstrate having dispersed therein an admixture that comprises aplasticizer and a lipophilic electrolyte having a lipophilic cationcomponent and a lipophilic anion component. Preferentially, one of thelipophilic cation component and the lipophilic anion component isselective for a specific polyion.

In another aspect of the invention, there is provided apolyion-selective membrane electrode for use in an electrochemical cell.In one embodiment, the polyion-selective membrane electrode comprises ahousing, a reference solution contained within the housing, and anelectrode operatively positioned within the housing such that theelectrode is in contact with the reference solution. Further, accordingto this embodiment, a polyion-selective membrane is disposed at one endof the housing. The membrane is in contact with the reference solutionwithin the housing, and the membrane is operatively positioned forcontacting a sample solution that is external to the housing. Themembrane comprises a lipophilic electrolyte having a lipophilic cationcomponent and a lipophilic anion component, wherein at least one of thelipophilic anion and lipophilic cation components is selective for aspecific polyion.

In another aspect of the present invention, there is provided a methodof measuring the concentration of a polyion species in a samplesolution. The method according to this aspect of the invention iscapable of electrochemically controlled, reversible transport of thepolyion species across a membrane. Therefore, the method is useful forcontinuous measurement of the concentration of a polyion species in asample solution, such as a biological sample.

According to one embodiment of the method, there is provided a samplesolution having therein a polyion species. Preferentially, the samplesolution further comprises a background electrolyte. The solution iscontacted with a reference electrode and a membrane electrode that areelectrically connected. The membrane of the membrane electrode iscomprised of a lipophilic electrolyte having a lipophilic cationcomponent and a lipophilic anion component, at least one of thelipophilic anion and lipophilic cation components being selective forthe polyion species. When the sample solution is in contact with theelectrodes, an external current pulse is applied to a circuit comprisingthe membrane electrode and the sample solution, the applied currentdriving transport of the polyion species from the sample solution intothe membrane. Preferably, the external current pulse is of fixedduration. A measurement of the potentiometric response between themembrane electrode and the reference electrode can be taken during thecurrent pulse. The concentration of the polyion species is then capableof being calculated as a function of the potentiometric response.

In another embodiment of this aspect of the present invention, themethod further comprises applying an external electrode potential to themembrane electrode and the reference electrode, thereby drivingtransport of the polyion species from the membrane. In this embodiment,the method allows for a reversible sensor, wherein the polyion isback-extracted, the membrane thus being reconditioned for further use.

In still another embodiment of the invention according to this aspect, amethod of measuring the concentration of a polyion species in a samplesolution is provided. The method comprises the following steps:

a) providing a sample solution comprising a polyion species and abackground electrolyte;

b) providing an electrochemical cell apparatus comprising i) apolyion-selective membrane electrode comprising a membrane thatcomprises a lipophilic electrolyte having a lipophilic cation componentand a lipophilic anion component, wherein at least one of the lipophilicanion and lipophilic cation components is selective for a specificpolyion, ii) a reference electrode electrically connected to themembrane electrode, iii) a counter electrode electrically connected tothe membrane electrode, iv) an electrochemical instrument operativelyconnected to the electrodes, and v) a controller device in communicationwith the electrochemical instrument;

c) contacting the sample solution with the electrodes of theelectrochemical cell apparatus;

d) applying an external current pulse of fixed duration to a circuitcomprising the membrane electrode, the counter electrode, and the samplesolution;

e) measuring a potentiometric response during the current pulse;

f) calculating the concentration of the polyion species as a function ofthe potentiometric response; and

g) applying an external electrode potential to the membrane electrodeand the reference electrode, thereby driving transport of the polyionspecies from the membrane. In a preferred embodiment, steps d) throughg) are repeated to obtain one or more additional measurements of theconcentration of the polyion species.

According to another aspect of the present invention, there is providedan electrochemical cell apparatus. The apparatus is useful for measuringthe concentration of a polyion in a sample solution. The apparatusaccording to one embodiment comprises: a polyion-selective membraneelectrode comprising a membrane comprising a lipophilic electrolytehaving a lipophilic cation component and a lipophilic anion component,wherein at least one of the lipophilic anion and lipophilic cationcomponents is selective for a specific polyion; a reference electrodeelectrically connected to the membrane electrode; and an electrochemicalinstrument operatively connected to the membrane electrode and referenceelectrode.

In another embodiment according to this aspect of the invention, theelectrochemical cell apparatus further comprises a counter electrodeelectrically connected to the membrane electrode.

In yet another embodiment according to this aspect of the invention, theelectrochemical cell apparatus further comprises a controller device incommunication with the electrochemical instrument. In a preferredembodiment, the controller device is a computerized controller. Suchcontroller allows for partial or full automation of the electrochemicalcell apparatus.

In another aspect of the present invention, a sensor is disclosed. Thesensor comprises an electrode positioned within a housing. A membrane isdisposed at one end of the housing and in contact with a sample solutionexternal to the housing. The membrane detects a polyion concentrationwithin the sample solution, and a rate of decomposition of the polyionconcentration is directly proportional to an enzyme activity within thesample solution.

The electrode can be a Ag/AgCl electrode. The membrane can have asurface area of about 10 mm² to about 100 mm². Alternatively, themembrane can have a surface area of about 20 mm2 to about 50 mm2. Themembrane can have an average thickness of about 10 μm to about 1000 μm.Alternatively, the membrane can have an average thickness of about 20 μmto about 300 μm. The polyion concentration can be that of protamine. Theenzyme activity can be that of trypsin.

In another aspect of the present invention, a method of detecting apolyion concentration in a sample solution is disclosed. The methodcomprises the step of providing a sample solution. The method alsocomprises the step of contacting the sample solution with a membrane,wherein the membrane detects a polyion concentration within the samplesolution, and wherein a rate of decomposition of the polyionconcentration is directly proportional to an enzyme activity within thesample solution. The sample solution can comprise a biologicalcomponent. The sample solution can comprise blood.

In another aspect of the present invention, a reversible electrochemicalcell apparatus is disclosed. The cell apparatus comprises apolyion-selective membrane electrode, including a membrane. The membranecan detect a polyion concentration within the sample solution, and arate of decomposition of the polyion concentration is directlyproportional to an enzyme activity within the sample solution. The cellapparatus also includes means for applying a potential to clear themembrane. The means for applying a potential to clear the membrane canbe an external electrode potential applied between a reference electrodeand the polyion-selective membrane electrode.

In another aspect of the present invention, a reversible electrochemicalcell apparatus is disclosed. The cell apparatus includes apolyion-selective membrane electrode, including a membrane. The membranedetects a polyion concentration with a solution. The cell apparatus alsoincludes means for applying a potential between a reference electrodeand the polyion-selective membrane electrode to clear the membrane.

In another aspect of the present invention, a sensor is disclosed. Thesensor includes an electrode positioned within a housing. The sensoralso includes a membrane disposed at one end of the housing and incontact with a sample solution external to the housing. The membrane canmonitor an enzyme activity and a corresponding enzyme inhibitor activityin the sample solution.

The corresponding enzyme inhibitor activity can be one of a1-antiproteinase inhibitor, α2-macroglobulin, aprotinin, and a soybeaninhibitor. A potential decrease of the present invention can bedependent on the concentration of the corresponding enzyme inhibitor inthe sample solution.

In another aspect of the present invention, an immunoassay is disclosed.The immunoassay can detect analytes by employing one of a polyion and anenzyme as markers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an electrochemical cell apparatusincluding a polyion-selective membrane electrode according to apreferred embodiment of the present invention;

FIG. 2 is a chart illustrating electrode reproducibility uponalternating between 0.1 M NaCl and 0.1 M NaCl containing 10 mg/Lprotamine for (A) a polyion-selective membrane electrode according tothe present invention and (B) a prior art ion-selective electrode;

FIG. 3 is a chart illustrating current/time traces and potential/timetraces for the pulsed galvanostatic measurement of a solution of 0.1 MNaCl and a solution of 0.1 M NaCl and 10 mg/L protamine using a methodof measurement incorporating a polyion-selective membrane electrodeaccording to the present invention;

FIG. 4 is a chart illustrating current/time traces and potential/timetraces for the pulsed galvanostatic measurement of a solution of 0.1 MNaCl and a solution of 0.1 M NaCl and 50 mg/L protamine using a methodof measurement incorporating a polyion-selective membrane electrodeaccording to the present invention;

FIG. 5 is a chart illustrating calibration curves for protamine in 0.1 MNaCl using pulsed galvanostatic measurements and using (A) apolyion-selective membrane electrode according to the present inventionand (B) a prior art ion-selective electrode;

FIG. 6 is a chart illustrating the effect of stirring on the response ofa polyion-selective membrane electrode according to the presentinvention when applying a cathodic current of −2 μA in blank solution of0.1 M NaCl and in the presence of 10 mg/L of protamine;

FIG. 7 is a chart illustrating the influence of a sample pH on theresponse of a polyion-selective membrane electrode according to thepresent invention at a cathodic current of −2 μA without protamine(lower curve) and with 25 mg/L of protamine added (upper curve);

FIG. 8 is a chart illustrating calibration curves for apolyion-selective membrane electrode according to the present inventionin pure solutions of NaCl, KCl, MgCl₂, CaCl₂ and of protamine in a 0.1 MNaCl background electrolyte solution;

FIG. 9 provides two charts illustrating (A) calibration curves ofprotamine in the presence of different concentrations of supportingelectrolyte (0.01 M NaCl, 0.03 M NaCl, and 0.1 M NaCl) and (B) theeffect of KCl concentration on protamine calibration curves in 0.1 MNaCl with and without 0.01 M Kcl;

FIG. 10 is a chart illustrating amplitude-time behavior of potential forthe protamine titration in whole blood using a polyion-selectivemembrane electrode according to the present invention;

FIG. 11 is a chart illustrating (A) titration of whole blood samples,containing 0 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM concentrations ofheparin with 1 g/L of protamine solution using a polyion-selectivemembrane electrode according to the present invention and (B) acorresponding calibration curve for heparin-protamine titration in wholeblood using a polyion-selective membrane electrode according to thepresent invention;

FIG. 12 a is a chart illustrating the reaction rate of potential change(ΔE/Δt) immediately after the addition of trypsin, as a function oftrypsin concentration;

FIG. 12 b is a chart illustrating the reaction rate of protamineconcentration change (ΔC/Δt) immediately after the addition of trypsin,as a function of trypsin concentration;

FIG. 13 a is a chart illustrating the reaction rate of potential change(ΔE/Δt), immediately after the addition of trypsin and SI inhibitor, asa function of SI inhibitor concentration; and

FIG. 13 b is a chart illustrating the reaction rate of protamineconcentration change (ΔC/Δt) immediately after the addition of trypsinand SI inhibitor, as a function of SI inhibitor concentration.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention now will be described more fully hereinafter.However, this invention may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. As used in this specification andthe claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

The present invention provides a reversible polyion sensor that combinesthe process of mass transport limited polyion extraction duringmeasurement with subsequent back-extraction for reconditioning undersequential instrumental control. The invention uses a polyion-selectivemembrane comprising a lipophilic electrolyte. The instrumental controlof ion fluxes across the membrane allows one to repeatedly extract ionsinto the membrane and strip ions from the membrane, yielding highlyreproducible sensor responses. Further, the ability to rehabilitate thesensor membrane quickly, and during testing procedures, allows forcontinuous functioning to provide real-world values for polyionconcentrations in solution.

Potentiometric polyion-selective sensors previously known in the art arepassive sensors. Such sensors have membranes that contain lipophiliccation-exchanger molecules (generally described by the formula R⁻Na+)and an aqueous solution of an electrolyte. The phase boundary potentialbetween a sample and the membrane in such a sensor may be determinedaccording to equation (1): $\begin{matrix}{E_{PB} = {E^{0} + {\frac{RT}{F}\ln\frac{a_{Na}}{\left\lbrack {Na}^{+} \right\rbrack_{pb}}}}} & (1)\end{matrix}$wherein a_(Na) is the activity of sodium ions in the aqueous solution,[Na⁺] is the so-called free concentration of sodium ions at the phaseboundary of the membrane phase and E⁰ incorporates the free energy oftransfer for sodium from water to the membrane phase. The terms R and Tare the gas constant and the absolute temperature, respectively. In theabsence of protamine (or other polycation) in the sample, and byneglecting ion-pairing, the concentration of sodium ions in the membranephase is determined by the total concentration of the lipophiliccation-exchanger, R_(T), which can be calculated according to equation(2).[Na⁺]_(pb)=R_(T)   (2)

Consequently the membrane behaves like an ion-exchanger based sodiumelectrode and a Nernst response slope is expected.

If protamine is present in the aqueous solution, a strong flux of theprotamine cations occurs both to the surface and into the membranephase, forming two stagnant diffusion layers. Because the diffusion inthe stagnant layer of the aqueous phase is the rate-limiting step, aquasi-steady-state diffusion may be observed at the interface. Protaminecations displace the sodium cations from the membrane phase boundary.This ion-exchange process reduces the concentration of sodium ions inthe membrane phase and increases the observed potential, as calculatedin equation (1). This increase in observed potential can be accountedfor because the total concentration of cations in the system mustsatisfy the electroneutrality condition as described by equation (3)[Na⁺]_(pb)=R_(T) −z[PA ^(z+)]_(pb)   (3)where [PA^(z+)]_(pb) is the concentration of protamine with charge z inthe membrane phase boundary. That concentration can be formulated as afunction of the bulk protamine concentration on the basis of a pseudosteady-state flux consideration calculated according to equation (4),$\begin{matrix}{\left\lbrack {PA}^{Z +} \right\rbrack_{pb} = {\frac{D_{{aq},{PA}}\delta_{m}}{D_{m,{PA}}\delta_{aq}}c_{{PA},{bulk}}}} & (4)\end{matrix}$where D_(m), D_(aq), δ_(m) and δ_(aq), are diffusion coefficients ofprotamine in the membrane phase, aqueous solution, and resultingdiffusion layer thickness respectively. Equation (4) may now be insertedinto equation (3) and equation (1) to obtain the protamine response atlow concentrations. The resulting equation (5) is provided below.$\begin{matrix}{E_{PB} = {E^{0} + {\frac{RT}{F}\ln\frac{a_{Na}}{R_{T} - {\frac{{zD}_{{aq},{PA}}\delta_{m}}{D_{m,{PA}}\delta_{aq}}c_{{PA},{bulk}}}}}}} & (5)\end{matrix}$

As can be determined in the above calculations, if a_(Na) is fixed, thephase boundary potential of the membrane shows a direct response forprotamine. Accordingly, at high protamine concentration, the sodium ionsare quantitatively replaced from the membrane and a near-Nernstianresponse slope for protamine is expected. Such a quantitativereplacement also occurs with dilute protamine solutions but requiresprolonged exposure (ca. 24 h). The resulting response slope is too smallto be analytically useful.

Such spontaneous ion extraction sensors suffer from the problemspreviously described, such as signal drift upon prolonged use and thelimitation to a single use for most sensors. Accordingly, until thepresent invention, there was not an easy, reliable method forreconditioning the sensor allowing for continuous use.

In contrast to the passive potentiometric sensors described above thatrely on spontaneous ion exchange in the ion-extraction process, thepresent invention electrochemically induces ion extraction by applying aconstant current pulse. In order to prevent spontaneous extraction, themembrane contains a highly lipophilic electrolyte that can generally bedefined according to the formula R⁺ R⁻ and does not possess intrinsicion-exchange properties. This being the case, the initial concentrationsof protamine or sodium cations in the membrane bulk is assumed to bezero. An applied cathodic current i induces a net flux of cations J inthe direction of the membrane phase. To simplify the resulting equation,it can be assumed that only sodium and protamine ions can be extractedinto the membrane phase. Accordingly, the relationship between current iand the fluxes of sodium, J_(Na), and protamine, J_(PA), can becalculated according to equation (6).i=FAJ _(Na) +zFAJ _(PA)   (6)where A is the exposed membrane area. Assuming linear concentrationgradients for simplicity and recalling that the sodium concentration inthe membrane bulk is zero, the sodium flux can be related to theconcentration gradient across the organic phase boundary as follows inequation (7). $\begin{matrix}{J_{Na} = {- {\frac{D_{m,{Na}}}{\delta_{m}}\left\lbrack {Na}^{+} \right\rbrack}_{pb}}} & (7)\end{matrix}$

If no protamine is present, equation (6) and equation (7) may beinserted into equation (1) to give equation (8) shown below.$\begin{matrix}{E_{PB} = {E^{0} + {\frac{RT}{F}\ln\frac{{FAD}_{m,{Na}}}{i\quad\delta_{m}}a_{Na}}}} & (8)\end{matrix}$

A cathodic current pulse of fixed duration and magnitude, followed by apotentiostatic stripping pulse to keep the membrane bulk void of sodiumions, will give a near-Nemstian electrode slope. If protamine is presentin the sample solution, the protamine will effectively compete with thesodium ions in the extraction process. Equation (6) can be rewritten inanalogy to equation (7) as follows in equation (9). $\begin{matrix}{i = {{{- {FA}}{\frac{D_{m,{Na}}}{\delta_{m}}\left\lbrack {Na}^{+} \right\rbrack}_{pb}} - {{zFA}{\frac{D_{m,{PA}}}{\delta_{m}}\left\lbrack {PA}^{Z +} \right\rbrack}_{pb}}}} & (9)\end{matrix}$

Assuming now that the applied current imposes a flux that is alwayslarger than the flux that can be sustained by polycation diffusionalone, equation (4) is still valid and can be inserted into equation(9). As a result, the sodium flux, J_(Na), is decreased, which increasesthe potential according to equation (1). Inserting equation (4) intoequation (9), solving for [Na⁺]_(pb), and substituting into equation (1)therefore yields the predicted protamine response at low polyionconcentrations, which is provided below in equation (10).$\begin{matrix}{E_{PB} = {E^{0} + {\frac{RT}{F}\ln\frac{a_{Na}}{\frac{\delta_{m}}{D_{m,{Na}}}\left( {\frac{- i}{FA} - {z\frac{D_{{aq},{PA}}}{\delta_{aq}}c_{{PA},{bulk}}}} \right)}}}} & (10)\end{matrix}$

There are differences between equation (10) and the protamine responseshown in equation (5) for a potentiometric sensor as known in the priorart. Importantly, the diffusion layer thickness in the membrane phase isnow dictated galvanostatically, and potentiostatic membrane renewalbetween pulses assures repeatable δ_(m) values from pulse to pulse.While the embodiments provided in the present invention use a currentprimarily chosen to give a maximum potential range, the invention is notintended to be so limited. Accordingly, the magnitude of the appliedcurrent pulse can be used to adjust the measuring range for polyionresponse. Since the applied current, and not an ion-exchanger, dictatesthe extraction of sodium ions into a pulsed chronopotentiometricallycontrolled membrane, the diffusion coefficient in the membrane phasedoes not influence the protamine response range. This is in contrastwith the prior art potentiometric sensors governed by formula (5), wherea direct dependence between competitive extraction of sodium byprotamine and the membrane diffusion coefficients is known to exist.

With the above background theory in place, one can now easily see theadvantages provided by the present invention. In particular, the presentinvention provides a polyion-selective membrane for use in anelectrochemical cell. Further, the polyion-selective membrane can be anintegral part of an electrochemical cell electrode. Thepolyion-selective membrane and the membrane electrode can be used in areversible method of measuring the concentration of a polyion species ina sample solution.

The polyion-selective membranes of the present invention arecharacterized by comprising a lipophilic electrolyte having a lipophiliccation component and a lipophilic anion component, wherein at least oneof the lipophilic anion and lipophilic cation components is selectivefor a specific polyion. The term lipophilic is generally understood todescribe a species having an affinity for fat and having high lipidsolubility. Lipophilicity is a physicochemical property that describes apartitioning equilibrium of a particular species between water and animmiscible organic. Lipophilicity can further be described as theability of a species to dissolve in a lipid phase when an aqueous phaseis also present. This relationship (i.e. the partition coefficient) canbe defined as the equilibrium constant of the concentrations of thespecies in the two phases. The standard for comparison is generally a1-octanol/water partition coefficient. The partition coefficient can becalculated according to equation (11) shown below. $\begin{matrix}{P = \frac{\lbrack{molecule}\rbrack_{lipid}}{\lbrack{molecule}\rbrack_{water}}} & (11)\end{matrix}$

According to equation (11), molecules exhibiting a high lipophilicitywould be expected to show preference for solubility in a lipid ratherthan in water.

One functional test for determining lipophilicity is to place thecompound to be tested in a container holding a mixture of 50% water and50% lipid (such as 1-octanol). The compound of interest can be placed inthe container, and a mixing force can be applied to force distributionof the compound in both phases. The container can then be allowed torest such that the compound is allowed to come to a concentrationequilibrium between the phases. The concentration of the compound ineach phase can then be measured, and the concentrations can be used inequation (11) for determining lipophilicity.

Computer software can also be used to determine the lipophilicity of aspecies. One example of a computer program for determining lipophilicityis the ALOGPS program available online athttp://146.107.217.178/lab/alogps. Principles surrounding lipophilicityare also discussed by Bakker, E. and Pretsch, E., “Lipophilicity oftetraphenylborate derivatives as anionic sites in neutral carrier-basedsolvent polymeric membranes and the lifetime of correspondingion-selective electrochemical and optical sensors” Analytica ChimicaActa, 1995, 309, 7-17, which is incorporated herein by reference in itsentirety.

Generally, a compound with a calculated P value greater than 100,000 isconsidered to be highly lipophilic, and therefore useful according tothe present invention. Using compounds having even higher P values,however, can generally be expected to lead to sensors having increasedlifetimes. Accordingly, it is preferred that the lipophilic compoundsused according to the present invention have a P value of greater than100,000, more preferably, greater than 1,000,000, and most preferably,greater than 10,000,000.

Polyion-selective membranes known in the prior art include a lipophilicelectrolyte and a hydrophilic counter cation (i.e., R⁻Na⁺). According tothe present invention, the hydrophilic counterion is replaced by alipophilic counterion. Accordingly, the polyion-selective membrane ofthe present invention comprises a lipophilic electrolyte having alipophilic cation component and a lipophilic anion component (i.e. R⁻R⁺). By using two lipophilic electrolytes, the lipophilic counterion mayno longer spontaneously exchange with the polyion species being measuredin the sample. Preferably, one of the lipophilic anion and lipophiliccation components is selective for a specific polyion, facilitatingsensing of that polyion. Non-limiting examples of specific polyions forwhich sensing is desired include protamine, heparin, humic acids,carrageenans, deoxyribonucleic acids, ribonucleic acids, and otherpolyionic macromolecules.

In one embodiment of the present invention, the lipophilic anioncomponent of the lipophilic electrolyte is selective for protamine.Protamine selectivity of the lipophilic anion component is dependantupon the functional groups of the anion. Protamine contains basicguanidinium groups (i.e., arginine residues). Therefore, in order to beselective for protamine, the lipophilic anion must include functionalgroups capable of forming ion pairs with the guanidinium groups ofprotamine. In a preferred embodiment, lipophilic anions havingcarboxylic (COOH), sulfonic (SO₃H), or sulfuric (OSO₃H) groups are usedfor protamine selectivity. In a particularly preferred embodiment, thelipophilic anion component of the lipophilic electrolyte is selectedfrom the group consisting of 1,3-dinonylnaphthalene-4-sulfonate,2,6-dinonylnaphthalene-4-sulfonate, dodecylbenzenesulfonate, and3,9-diethyl-6-tridecylsulfate. Chemical formulas for these compounds areshown below.

As described above, when the hydrophilic counterion of known membraneelectrolyte materials is replaced with a second lipophilic electrolyte,the spontaneous extraction of ions from a sample into thepolyion-selective membrane is prevented. Generally, the hydrophiliccounterion is sodium, since sodium is the most abundant counterionpresent in sample solutions. The sodium ions are replaced with alipophilic counterion by chemical synthesis. When the polyion-selectiveanion is selective for protamine, it is expected that any lipophilicquaternary ammonium cation with an alkyl side arm chain length of about4 to about 16 would be a suitable counterion.

In a preferred embodiment, the hydrophilic counterion paired with theprotamine-selective lipophilic anion is a cation selected from the groupconsisting of tetradodecylammonium, tridodecylmethylammonium, anddodecyltrimethylammonium. The chemical formulas for these cations areshown below.

According to the above description of lipophilic ions, it is possible toselect a combination of a lipophilic anion selective for protamine and alipophilic counter cation to suppress the spontaneous ion exchange withthe sample solution. Therefore, a protamine-selective lipophilicelectrolyte for use according to the present invention could be selectedfrom any of the possible combinations of the protamine-selective anionsand the counter cations provided above. According to one preferredembodiment, the lipophilic electrolyte for use in selectively extractingprotamine from a solution is tetradodecylammonium1,3-dinonylnaphthalene-4-sulfonate (TDDA-DNNS).

In another embodiment according to the present invention, the lipophilicelectrolyte comprises a lipophilic cation component that is selectivefor heparin. Heparin selectivity of the cation component is dependantupon the functional groups of the compound. Heparin contains sulfonicand carboxylic groups. Therefore, in order to be selective for heparin,a suitable cation must contain one or more groups that can form ionpairs with the sulfonic and carboxylic groups of the heparin.Particularly useful in providing heparin selectivity are guanidiniumgroups. Further, heparin extracted from a sample into an organic sensingphase (such as a membrane according to the present invention) isstabilized by stacking via long aliphatic side chains or aromatic ringsof neighboring cations. Therefore, cations with high lipophilicity canbe prepared by attaching one or more guanidinium groups to an aliphaticchain having a chain length of about 4 to about 18 carbon atoms and/orsuitable aromatic functionalities. In a preferred embodiment, thelipophilic cation component of the lipophilic electrolyte is selectedfrom the group consisting of dodecylguanidinium andN,N′-1,10-decanediylbis(guanidinium). The chemical formulas for thesecations are shown below.

Again, when the hydrophilic counterion is replaced with a secondlipophilic electrolyte, the spontaneous extraction of ions from a sampleis prevented. Generally, the most abundant anion in test solutions,chloride, is replaced via chemical synthesis with a lipophilic anion.One group of anions useful as counterions according to this embodimentare tetraphenylborate derivatives, such as the three borates providedbelow.

Another suitable group of anions is lipophilic (perhalogenated oralkylated) dodecacarboranes. Dodecacarboranes are based upon theicosahedral carborane anion which, in its fully unsubstituted form, hasthe chemical formula CB₁₁H₁₂ ⁻. Halogenated dodecacarboranes, such as1-H—CB₁₁Cl₁₁, 1-H—CB₁₁Br₁₁, and 1-H—CB₁₁I₁₁, are especially usefulaccording to the present invention and are more fully described byPeper, S. et al., “Ion-pairing. Ability, Chemical Stability, andSelectivity Behavior of Halogenated Dodecacarborane Cation Exchangers inNeutral Carrier-Based Ion-Selective Electrodes,” Analytical Chemistry,(2003) 75(9), 2131-2139, which is incorporated by reference in itsentirety. Also useful according to the present invention are alkylateddodecacarboranes, wherein the halogen groups as described above arereplaced various alkyl groups. In a preferred embodiment, thehydrophilic counterion paired with the heparin-selective lipophiliccation is a tetrakis(p-chlorophenyl)borate anion.

According to the above description of lipophilic ions, it is possible toselect a combination of a lipophilic cation selective for heparin and alipophilic counter anion to suppress the spontaneous ion exchange withthe sample solution. Therefore, a heparin-selective lipophilicelectrolyte for use according to the present invention could be selectedfrom any of the possible combinations of the heparin-selective cationsand the counter anions provided above. According to one preferredembodiment, the lipophilic electrolyte for use in selectively extractingheparin from a solution is dodecylguanidiniumtetrakist(p-chlorophenyl)borate (DDG-TCIPB).

Using the principles outlined above, it is also possible to determinelipophilic electrolytes having a lipophilic cation or lipophilic anioncomponent that is selective for a specific polyion other than protamineor heparin. Accordingly, membranes incorporating such lipophilicelectrolytes are also encompassed by the present invention.

The amount of lipophilic electrolyte present in the membrane accordingto the present invention can vary depending upon the physical propertiesof the membrane, which can limit the solubility of a salt in themembrane. Preferably, the lipophilic electrolyte is present at about 1to about 15 weight percent based upon the total weight of the membrane.More preferably, the lipophilic electrolyte is present at about 5 toabout 12 weight percent based upon the total weight of the membrane. Inone preferred embodiment, the lipophilic electrolyte is present at about10 weight percent based upon the total weight of the membrane.

In addition to the lipophilic electrolyte, the membrane according to thepresent invention can further comprise one or more plasticizers. Theplasticizer facilitates mixture homogeneity and also helps control theflux of the polyion from the sample solution to the surface of themembrane and into the bulk of the membrane. Various plasticizers can beused in the membrane of the present invention including, but not limitedto, plasticizers selected from the group consisting of 2-nitrophenyloctyl ether, dioctyl phthalate, dioctyl sebacate, dioctyl adipate,dibutyl sebacate, dibutyl phthalate, 1-decanol, 5-phenyl-1-pentanol,tetraundecyl benzhydrol 3,3′,4,4′-tetracarboxylate, benzyl ether,dioctylphenyl phosphonate, tris (2-ethylhexyl) phosphate, and2-nitrophenyl octyl ether. In a preferred embodiment according to thepresent invention, the plasticizer used in the membrane is 2-nitrophenyloctyl ether (NPOE).

It is generally preferred that that membrane of the present invention,in addition to the lipophilic electrolyte and the plasticizer, furthercomprise a substrate material to function as the bulk forming materialof the membrane. Multiple substrates for use in forming a permeablemembrane are known to those of skill in the art, and it is intended thatthe present invention encompass all such substrates.

In one embodiment, the substrate material is a polymeric film-formingmaterial. The polymeric film-forming material according to thisembodiment can be any polymeric material chemically compatible with thelipophilic electrolyte and the plasticizer. Further, the polymericmaterial should be capable of being formed into a film, such as throughsolvent casting. Polymeric materials useful according to the presentinvention include, as non-limiting examples, polyvinyl chloride,polyurethane, cellulose triacetate, polyvinyl alcohol, silicone rubber,and copolymers and terpolymers thereof. In one preferred embodiment, thepolymeric film-forming material is polyvinyl chloride.

In one embodiment of the invention, the polyion-selective membranecomprises a lipophilic electrolyte in an amount of about 1 to about 15weight percent. The membrane according to this embodiment furthercomprises about 28 to about 49.5 weight percent of a polymericfilm-forming material and about 42.5 to about 66 weight percent of aplasticizer (all weights being based upon the total weight of themembrane). Preferentially, the polymeric film-forming material and theplasticizer are present in a ratio of about 1:1 to about 1:2 by weight.

In a preferred embodiment, the polyion-selective membrane comprisesabout 10 weight percent tetradodecylammonium1,3-dinonylnaphthalene-4-sul-fonate, about 30 weight percent polyvinylchloride, and about 60 weight percent 2-nitrophenyl octyl ether (allweights being based upon the total weight of the membrane).

A polyion-selective membrane according to one of the above embodimentscan be prepared by solvent casting with an organic solvent, such astetrahydrofuran (THF), suitable for casting into a thin film.Preferentially, the polymeric film-forming material, the plasticizer,and the lipophilic electrolyte are prepared as a homogeneous solution inthe solvent. The solution can then be cast into a thin film. Onceprepared as a thin film, the membrane can be cut to any specified sizefor later use in a polyion sensor. Rather than being formed into a thinfilm, the membrane solution can be applied to a substrate, such as anelectrode, and allowed to dry on the electrode, thereby forming a filmdirectly on the electrode.

According to one embodiment of the present invention, at least one ofthe lipophilic anion component and the lipophilic cation component canbe covalently attached to the backbone structure of the polymericfilm-forming material. For example, the anion component can be attachedto the polymer chain through copolymerization through vinyl grouplinkage or some other suitable form of chemical reaction. Further, thelipophilic cation or anion component capable of attachment to thepolymer structure can be a polyion-selective component or a counterioncomponent.

In another embodiment, the substrate material is a microporoushydrophobic substrate. According to this embodiment, the plasticizer andthe lipophilic electrolyte are formed into an admixture and thendispersed onto a microporous hydrophobic substrate, wherein theadmixture of the plasticizer and the lipophilic electrolyte are taken upinto the pores of the substrate and allowed to cure. The microporoushydrophobic substrate with the plasticizer and lipophilic electrolytedispersed therein can then be processed for use in a polyion sensor. Themicroporous hydrophobic substrate according to one embodiment of theinvention, can be selected from the group consisting of polyethylene,polypropylene, nylon, polyvinylidene fluoride, polycarbonate,polytetrafluoroethylene, acrylic copolymer, polyether sulfone, andcopolymers and terpolymers thereof. According to one preferredembodiment, the microporous hydrophobic substrate is polyethylene.Particularly preferred as the microporous hydrophobic substrate areCelgard® membranes, available from Celgard, Inc., Charlotte, N.C.Celgard®. membranes are polyethylene-based membranes available as flatsheet membranes and hollow fiber membranes.

In one preferred embodiment of the present invention, thepolyion-selective membrane comprises a microporous hydrophobic substratethat has been contacted with an admixture comprising about 1 to about 15weight percent of a lipophilic electrolyte comprising a lipophiliccation component and a lipophilic anion component and about 85 to about99 weight percent of a plasticizer, based on the total weight of themixture.

The present invention further provides a polyion-selective membraneelectrode that is useful in an electrochemical cell. In one embodimentof the invention, the polyion-selective membrane electrode comprises ahousing, a reference solution contained within the housing, and anelectrode operatively positioned within the housing such that theelectrode is in contact with the reference solution. Further, accordingto this embodiment, a polyion-selective membrane is disposed at one endof the housing. The membrane is in contact with the reference solutionwithin the housing, and the membrane is operatively positioned forcontacting a sample solution that is external to the housing. As notedabove, the membrane comprises a lipophilic electrolyte having alipophilic cation component and a lipophilic anion component, wherein atleast one of the lipophilic anion and lipophilic cation components isselective for a specific polyion.

Any standard electrode could be used according to this embodiment of theinvention, so long as the electrode is capable of incorporating apolyion-selective membrane as described above. In a particularlypreferred embodiment of the present invention, the membrane electrodecomprises a polyion-selective membrane incorporated into a standardelectrode, such as a Philips electrode body (IS-561, Glasblserei Moller,Zurich, Switzerland).

The reference solution used in the electrode housing can be anyelectrolyte solution generally known to one of skill in the art as beinguseful. In one preferred embodiment, the electrolyte solution is asodium chloride solution, in particular, a 1 M NaCl solution. Further,the electrode itself can be any type of electrode capable of use inelectrochemical cells of potential and current values as describedbelow. Particularly useful is a Ag/AgCl electrode.

The polyion-selective membrane incorporated into the membrane electrodeis, according to one preferred embodiment, selective for protamine.Preferably, according to this embodiment, the lipophilic electrolyteused in the polyion-selective membrane is TDDA-DNNS.

When used with a membrane electrode, it is preferable that thepolyion-selective membrane have a surface area of about 10 mm² to about100 mm². More preferable is a surface area of about 20 mm² to about 50mm². To achieve such surface areas, a thin film can be prepared, asdescribed above, and the thin-film cut to the desired size, such as witha cork borer, for association with the electrode. It is furtherpreferable that the polyion-selective membrane have an average thicknessof about 10 μm to about 1000 μm, more preferably about 20 μm to about300 μm.

The present invention is further directed to an electrochemical cellapparatus. In one embodiment, the electrochemical cell apparatuscomprises a polyion-selective membrane electrode as previouslydescribed, a reference electrode electrically connected to the membraneelectrode, and an electrochemical instrument operatively connected tothe membrane electrode and the reference electrode.

One embodiment of an electrochemical cell apparatus according to thepresent invention is provided in FIG. 1, which shows an electrochemicalcell apparatus 5 useful for measurement of a polyion species in asample. FIG. 1 shows a polyion-selective membrane electrode 10, areference electrode 30, and a counter electrode 50 operativelypositioned in a testing sample container 60 having disposed therein asample solution 65. The membrane electrode 10 comprises an electrodehousing 15, a reference solution 17, and a reference electrode wire 21.Disposed at one end of the electrode housing 15 is a polyion-selectivemembrane 25 according to the present invention. The reference electrode30, as shown in FIG. 1, is a double-junction electrode, although othertypes of reference electrodes could be used without departing from theinvention. The reference electrode 30 includes an outer housing 33, aninner housing, 36, an outer housing reference solution 39, an innerhousing reference solution 41, and a reference electrode wire 43.

As seen in FIG. 1, the polyion-selective membrane electrode 10, thereference electrode 30, and the counter electrode 50 are eachoperatively connected to an electrochemical instrument 75, which isfurther in communication with a controller device 90. Theelectrochemical instrument 75 is preferably a galvanostat-potentiostat.Accordingly, the electrochemical instrument is capable of controllingthe current through the electrochemical cell at a preset value and isalso capable of controlling the electrical potential between the workingelectrode (e.g. the polyion-selective membrane electrode 10) and thereference electrode 30 at a preset value. In performing the latterfunction, the electrochemical instrument 75 is capable of forcingwhatever current is necessary between the working electrode (e.g. thepolyion-selective membrane electrode 10) and the counter electrode 50 tokeep the desired potential. In one particularly preferred embodiment,the electrochemical instrument 75 is a bipotentiostat, such as an AFCBP1Bipotentiostat available from Pine Instruments (Grove City, Pa.).

The controller device 90, as shown in FIG. 1, is preferably a computercapable of carrying out an algorithm designed to automatically regulatethe function of the electrochemical instrument 75 in controllingcurrent, potential, or electrochemical activity desirable. Thecontroller device 90 is also preferably capable of collecting data fromthe electrochemical instrument 75 and displaying the data visually tothe user and/or storing the data. Of course, it is understood that boththe electrochemical instrument 75 and the controller device 90 in FIG. 1would be connected to a power supply (not shown). The present inventionis further directed to a method of measuring the concentration of apolyion species in a sample solution. The method generally comprises thefollowing steps: a) providing a sample solution comprising a polyionspecies and a background electrolyte; b) contacting the sample solutionwith a polyion-selective membrane electrode having a membrane comprisinga lipophilic electrolyte having a lipophilic cation component and alipophilic anion component, wherein at least one of the lipophilic anionand lipophilic cation components is selective for the polyion species;c) contacting the sample with a reference electrode, thepolyion-selective membrane electrode and the reference electrode beingelectrically connected; d) applying an external current pulse of fixedduration to a circuit comprising the polyion-selective membraneelectrode and the sample solution, thereby driving transport of thepolyion species from the sample solution into the membrane; e) measuringa potentiometric response during the current pulse between thepolyion-selective membrane electrode and the reference electrode; and f)calculating the concentration of the polyion species as a function ofthe potentiometric response.

The external current pulse of fixed duration is preferentially appliedfor a duration of about 0.1 seconds to about 2 seconds. It is generallyunnecessary to measure the potentiometric response for the full durationof the applied current pulse. Rather, it is preferred to measure thepotentiometric response for only a portion of the duration of theapplied external current pulse. In an especially preferred embodiment,the potentiometric response is measure during the last about 100milliseconds of the fixed duration of the external current pulse.

The value of the potential measured in the above method depends upon thetype of polyion present in the sample. For example, if a cation, such asprotamine, is present, a cathodic current (negative) is applied to thecell. When the current is applied, the measured potential becomes morenegative. When an anion, such as heparin, is present, an anodic current(positive) is applied to the cell. When the current is applied, themeasured potential becomes more positive.

While the circuit to which the external current is applied generallycomprises the polyion-selective membrane electrode and the samplesolution, the circuit will also comprise one or more further componentsof the electrochemical cell. For example, in one embodiment according tothe invention, the circuit further comprises a counter electrode. Thisembodiment would encompass electrochemical systems conventionallyreferred to as “three-electrode” electrochemical cells. Additionally, inanother embodiment, the circuit further comprises a reference electrode.This embodiment encompasses electrochemical systems conventionallyreferred to as “two-electrode” electrochemical cells. Three-electrodesystems are typically preferred to avoid degradation of the referenceelectrode that can occur when an external current is applied to suchelectrodes.

The absolute value of the potential measured in the above method willgenerally be expected to decrease with time due to the steadilyincreasing diffusion layer thickness in the membrane. As describedabove, when testing for the presence of a poly cation, such asprotamine, a cathodic current is applied and a negative potential isobserved. When protamine (or another poly cation) is present in thesample, the potential measured is significantly more positive than ifthe poly cation is not present. Conversely, when testing for thepresence of a poly anion, an anodic current is applied and the observedpotential is positive. If heparin (or another poly anion) is present inthe sample, the measured potential would be expected to be significantlymore negative than if the poly anion is not present. In both cases, themove toward a more positive or more negative charge is indicative ofpolyion extraction from the sample solution into the membrane. Aftersufficient time, the measurement would begin to fail due to theaccumulation of the polyion in the membrane.

In a preferred embodiment of the present invention, the membrane isrehabilitated. According to this embodiment, the above method furthercomprises applying an external electrode potential to thepolyion-selective membrane electrode and the reference electrode,thereby driving transport of the polyion species from the membrane. Oncethe membrane has been effectively stripped of the polyions, thepolyion-selective membrane electrode can be used again for measurementof the polyions in the sample solution. Continuous, reversible detectionof a polyion species becomes possible by repeatedly applying the pulsesequence comprising an external current pulse followed by an externalpotential pulse.

It is preferable that the external electrode potential that is appliedto strip the polyions from the membrane is a baseline potential. Thevalue of the baseline potential can vary depending upon the symmetry ofthe electrochemical cell. For example, in one embodiment, the membraneelectrode and the reference electrode use identical electrodes and haveinner reference solutions that are similar in composition to the samplesolution. In such a preferred embodiment, the baseline potential is 0 V.Additional embodiments are also envisioned, wherein the electrodesexhibit less symmetry in varying degrees. In these additionalembodiments, the baseline potential would be expected to vary from 0 V.The optimal baseline potential may be determined by disconnecting theelectrochemical instrument (see FIG. 1), and replacing it with a highimpedance voltmeter to measure the zero current potential between themembrane electrode and the reference electrode.

In order to effectively strip the polyions from the membrane, theexternal potential is preferably applied for a duration of time that isabout 10 to about 20 times longer than the fixed duration of theexternal current pulse.

Further embodiments of the present invention are more distinctlydescribed according to the following experimental examples.

Another aspect of the present invention is the detection of polyions,such as protamine, in a sample solution based on their directrelationship to an enzyme activity, such as trypsin. Enzyme activity canbe detected if the enzyme is able to cleave polyions into shorterpieces, since these short pieces are generally not well detected bypolyion sensors. Electrochemical detection, like the one disclosed inthe present invention, has advantages over conventionalspectrophotometric methods for monitoring enzyme activity innontransparent, colored, or turbid samples.

One embodiment of this aspect of the present invention is the use oftrypsin with protamine. Trypsin is an enzyme that cleaves proteins atthe carboxyl side of the basic amino acids lysine and arginine. Sinceprotamine is rich in arginine residues, trypsin will cleave protamine atthese junctions. The initial rate of protamine decomposition was foundto be directly proportional to trypsin activity when monitored using theelectrochemical apparatus and protamine sensor disclosed in the presentinvention. The rate of change of the potential of the sensor signal as afunction of time is the digestion of the protamine by the trypsin. Thisrelationship can be extended to a variety of polyions and theircorresponding enzymes.

Another embodiment of the present invention is application of apotential to clear the membrane to reuse it in the monitoring of enzymeactivity, polyion concentration, and enzyme inhibitor activity. Itshould be noted that other biological and chemical activities can bemonitored using the present invention.

In addition, the present invention can be used to monitor enzymeinhibitor activity in a sample solution. An enzyme inhibitor binds to anenzyme and effectively decreases the rate of reaction of the enzyme. Theinitial potential decrease upon addition of the mixture of enzyme andenzyme inhibitor is dependent on the concentration of enzyme inhibitorincluded in the sample solution.

One embodiment of this aspect of the present invention is for the enzymetrypsin and one of the following corresponding enzyme inhibitors:α1-antiproteinase inhibitor, α2-macroglobulin, aprotinin, and soybeaninhibitor.

In yet another aspect of the present invention, potentiometric polyionsensitive electrodes can find application in the non-separationimmunoassays. The immunoassay can employ labeled polyions or relatedenzymes as markers to detect analytes that can serve as a label throughthe competitive bidding of free analytes and marked analytes withantibodies.

Experimental

The present invention is more fully illustrated by the followingexamples, which are set forth to illustrate the present invention andare not to be construed as limiting thereof. Unless otherwise indicated,all percentages refer to percentages by weight based on the total weightof the polyion-selective membrane.

EXAMPLE 1 Preparation of Protamine-Selective Membrane

The ability of a sensor incorporating a polyion-selective membraneaccording to the present invention to be used in an electrochemical cellwas tested. A polycation-selective membrane was formulated, particularlyto be selective for the polycation protamine. The membrane wasformulated with 10 weight percent TDDA-DNNS in a 2:1 weight ratiomixture of 2-nitrophenyl octyl ether and polyvinyl chloride. Themembrane was prepared by solvent casting with THF as the solvent. Themixture was allowed to dry into a film, and a protamine-selectivemembrane of about 200 μm thickness was prepared. The membrane was cutwith a cork borer having a diameter of 6 mm to prepare membranes forincorporation into electrodes.

EXAMPLE 2 Preparation of Protamine-Selective Membrane Electrodes

The protamine-selective membranes prepared in Example 1 wereincorporated into electrodes. The electrode comprised a Philipselectrode body (IS-561), an inner reference solution of 0.1 M NaCl, andan electrode wire of Ag/AgCl. The protamine-selective membraneelectrodes were conditioned overnight before experimental use in asolution identical to the inner reference solution.

A set of 10 identical electrodes were prepared as described above andtested for consistency in a 0.1 M NaCl solution prior to actualexperimental use. Testing showed an inter-electrode variability of +/−7mV (standard deviation) at a given current in the range of 0 to −10 μA.

The membrane electrodes were also tested to evaluate reversibility. Themembrane was repeatedly exposed to two separate solutions, onecontaining 0.1 M NaCl, and one containing 0.1 M NaCl and 10 mg/Lprotamine. The same test was also performed using a prior artion-selective electrode. The results of the test are shown in FIG. 2,wherein the protamine-selective electrode membrane of the presentinvention is illustrated in curve A and the prior art electrode isillustrated in curve B. As can be seen in both curves, a greaterpotential is observed when protamine is present. In curve A, thepotential measurements were reproducible with a variation of +/−1 mV. Incurve B, however shows variation of greater than 50 mV in as little as 5cycles.

EXAMPLE 3 Chronopotentiometric Responses for Samples with and withoutProtamine

A chronopotentiogram in 0.1 M NaCl with and without protamine wasprepared. An electrochemical cell, such as that shown in FIG. 1, was setup using a protamine-selective membrane electrode as described inExample 2. The reference electrode was a double junction Ag/AgClelectrode with a 1 M LiOAc bridge electrolyte. The counter electrode wasa platinum wire.

The voltammetric experiments were performed with an AFCBP1Bipotentiostat (Pine Inst., Grove City, Pa.) controlled by aPCI-MIO-16E4 interface board and LabVIEW 5.0 Software (NationalInstruments, Austin, Tex.) on a Macintosh computer. Prior to theexperiment the operation of the first electrode output of thebipotentiostat (K1) was switched to current control with potentiostaticcontrol of output of the second working electrode (K2). In order toapply the current pulse, the working electrode was connected to the K1output via an analog switch controlled by external software. When thebaseline potential between current pulses was applied the workingelectrode was connected to the K2 output.

During the chronopotentiometric experiments, each applied constantcurrent pulse of −3 μA (1 s duration) was followed by a constantpotential pulse at 0 V (10 s duration). Sampled potentials, whichrepresented the sensor response, were obtained as the average valueduring last 100 ms of each current pulse. All experiments were conductedat laboratory ambient temperature (21.5±0.5° C.). Confidence intervalswere computed at the 95% level.

The experiment was run in two samples. The first sample contained only0.1 M NaCl, while the second sample contained 0.1 M NaCl and protamine(PA) at a concentration of 10 mg/L. The applied cathodic current of −3μA lead to extraction of protamine into the membrane, and the observedpotential is significantly different for the sample with protamine ascompared to the sample without protamine. A current-time trace andpotential-time trace for the chronopotentiometric experiment is providedin FIG. 3.

EXAMPLE 4 Chronopotentiometric Responses for Samples with and withoutProtamine at Increased Levels

A second chronopotentiogram in 0.1 M NaCl with and without protamine wasprepared using the same experimental set up as provided in Example 2.During the chronopotentiometric experiments, each applied constantcurrent pulse of −2 μA (1 s duration) was followed by a constantpotential pulse at 0 V (15 s duration). Sampled potentials, whichrepresented the sensor response, were obtained as the average valueduring last 100 ms of each current pulse. All experiments were conductedat laboratory ambient temperature (21.5±0.5° C.). Confidence intervalswere computed at the 95% level.

The experiment was again run in two samples. The first sample containedonly 0.1 M NaCl, while the second sample contained 0.1 M NaCl andprotamine at a concentration of 50 mg/L. The applied cathodic current of−3 μA lead to extraction of protamine into the membrane, and theobserved potential is significantly different for the sample withprotamine as compared to the sample without protamine. A current-timetrace and potential-time trace for the chronopotentiometric experimentis provided in FIG. 4.

During the potentiostatic resting pulse, the backdiffusion of the ionsfrom the membrane can be observed. This diffusion is slower whenprotamine is present in the sample, indicating a difference in thediffusion behavior between sodium and protamine ions. When the currentwas integrated over the entire resting pulse of 15 s, the calculatedcharge corresponded to 90% of the applied charge during the currentpulse.

EXAMPLE 5 Calibration Curves for Protamine Comparing Protamine-SelectiveMembrane Electrode of the Present Invention with Prior ArtPolyion-Selective Membranes

Continuous, reversible detection of protamine becomes possible byrepeatedly applying the pulse sequence as illustrated in FIG. 3 and FIG.4 and by sampling the potential reading at the end of each currentpulse. Accordingly, it is possible to obtain a protamine calibrationcurve.

Time traces for protamine calibration curves in 0.1 M NaCl were obtainedusing the methods described above in Examples 3 and 4. Curves wereobtained using a protamine-selective membrane electrode as described inExample 2 and a prior art ion-selective electrode. A comparison of thetwo curves is provided in FIG. 5, wherein the curve obtained using theprotamine-selective membrane electrode of the present invention isillustrated in curve A and the prior art electrode is illustrated incurve B. The strong potential drift observed in curve B originates fromthe poor control of the diffusion layer thickness on the membrane side.Logarithmic protamine concentrations (in mg/L) are indicated on thetraces.

EXAMPLE 6 Effect of Stirring on Sensor Response

With prior art potentiometric polyion sensors, the observed potentialsare known to be strongly influenced by the rate of sample stirring,which alters the aqueous diffusion layer and hence the polyion flux tothe membrane. In fact, recent work has confirmed a clear relationshipbetween measuring range and rotation speed in a rotating electrodesetup. To examine how stirring may affect the response of apolyion-selective membrane electrode according to the present inventionin a galvanostatic pulse experiment, the potential was measured inunstirred solution and at a stirring rate of 100 rpm. A comparison ofthe two is provided in FIG. 6.

In contrast to potentiometric results with a heparin responsivemembrane, where sudden stoppage of sample stirring caused the potentialchange of approximately 20 mV, the response of the pulse galvanostaticsensor of the present invention does not show significant influence onstirring rate. Potential difference between stirred and unstirred sampledoes not exceed 2-3 Mv.

EXAMPLE 7 Effect of pH on Sensor Response

Although the protamine sensor is intended to work in whole blood at thephysiological pH of 7.4, the influence of pH on the sensor response wasalso examined. FIG. 7 provides observed potentials at a cathodic currentof −2 μA. The lower trace is the observed potential with a blanksolution comprising 0.1 M NaCl, 6.6 mmol citric acid, 11 mmol boricacid, and 10 mmol phosphoric acid, with the pH adjusted using 1 M NaOH.The upper trace is the observed potential for the same solution with 25mg/L of protamine in the sample. Owing to the high protamineconcentration, the difference between the two potentials may be regardedas the maximum sensor response, or potential window, in 0.1 M NaCl.

EXAMPLE 8 Membrane Selectivity

The selectivity of the membrane was determined at pH 7.4 by recordingseparate calibration curves for the chloride salts of sodium, potassium,calcium and magnesium. Curves of the potential versus the log of theconcentration of the salt are provided in FIG. 8. The resultingselectivity coefficients are in good agreement with those reportedpreviously for DNNS based ISE membranes without additional ionophore.All slopes in the concentration range of 0.001 M-0.1 M were found to beslightly super-Nemstian (70-72 mV), which biases the selectivitycoefficients to some extent. The slopes may likely be explained by thecontribution of ion migration at the membrane interface on the basis ofthe Nemst-Plank equation, which has not yet been considered in thesimplified theoretical model. The abrupt potential jump around 10⁴ Moriginates from depletion processes at the membrane surface. A protaminecalibration curve in 0.1 M NaCl is also shown in FIG. 8. The higherpotential readings demonstrate a strong preference of this membrane forprotamine over all other tested cations.

EXAMPLE 9 Effect of Background Electrolyte Concentration

The background electrolyte concentration is expected to influence theprotamine response curve because the response principle is based on acompetitive extraction between the polyion and sodium ions. A lowersodium background concentration, for instance, is expected to give alarger potential range for the protamine response (see equation 10), andmay also lead to a shift of the response to lower protamineconcentrations (equation 10). FIG. 9A shows experimental protaminecalibration curves in the presence of three sodium chlorideconcentrations, 10 mM, 30 mM, and 100 mM. The protamine potential rangedecreases with increasing NaCl concentrations.

The small influence of potassium on the protamine response isillustrated in FIG. 9B where two protamine calibration curves in 0.1 MNaCl with and without 10 mM KCl are shown. The maximum deviation of theresponse observed at low protamine concentration does indeed not exceed5 mV.

EXAMPLE 10 Calibration Curve for Protamine in Whole Blood

FIG. 10 illustrates a calibration curve for protamine in whole blood andthe corresponding potential-time trace for the calibration curve at thecathodic current of −2 μA. In whole blood the potential response rangewas found as about 60 mV, acceptably large for a practical determinationof protamine in whole blood samples. Standard deviations of potentialsincreased up to 1.5 mV in comparison with 0.7 mV observed in bufferedNaCl solutions. The results indicate that protamine concentrations aslow as 0.5 mg/L can be determined with the current pulsedchronopotentiometric sensor.

EXAMPLE 11 Titration of Whole Blood Samples

The experimental protocol can be used for determining heparin in bloodvia endpoint detection of a protamine titration, in analogy to previouswork with potentiometric sensors. Small aliquots of heparin stocksolution (2×10⁻⁵ M, 1.5 g/L) were added to whole blood samples in orderto obtain different model concentrations of heparin in the range of 0.25to 2 μM (0.6 to 4.5 kU/L), and titrated with 1 g/L protamine. Theresulting titration curves are represented in FIG. 11A.

Each point was calculated as an average of 10 consecutive potentialreadings, giving standard deviations no larger than 1.5 mV.Reproducibility was evaluated by repeating each titration 4 times,giving deviations of starting and ending potentials of up to 7 mV fromsample to sample, while the total change of potential during titrationsremained the same. Since each collection tube contained 7.2 mg of thepotassium salt of EDTA and the amount of blood collected in each tubevaried from 2 to 4 mL, most of the deviation may be attributed tovariations in the potassium concentration (see FIG. 9B).

The observed endpoints are plotted in FIG. 11B as a function of thewhole blood heparin concentration, and an expected linear relationshipwas found (correlation coefficient 0.995). The linear regression of thiscalibration curve yielded was determined as C_(Heparin)=V (6.6±0.4)×10⁻³M/L−0.6 μM.

EXAMPLE 12 Lifetime and Sensor Stability

The lifetime and stability of the sensor are important parameters,especially if measurement is conducted in physiological media.Continuous pulsed chronopotentiomeric measurement in pH buffered 0.1 MNaCl containing 10 mg/L protamine for 3 hours, with 1 minute measurementintervals, gave no visible potential drift and a maximum potentialvariation of 2 mV. For whole blood samples, the titration curves shownin FIG. 11B were obtained with the same sensor and the total time ofmeasurements in the blood exceeded 2.5 hours (for each point 10potential measurements were collected). After exposure to blood thesensors were placed in buffered 0.1 M NaCl and the baseline potentialwas found to return to the initial value (±5 mV for each sensor). Thelifetime of sensors, defined as the time where baseline potential shiftsdid not exceed 20 mV, was at least 2 weeks, with a 10 h total exposuretime to undiluted whole blood samples.

EXAMPLE 13 Preparation of the Protamine-Selective Membrane Electrodesand Detection of Trypsin Activity and Inhibitor Activity

The ion-selective membranes (200 μm thick) contained PVC and o-NPOE, 1:2by weight and 5 wt % lipophilic salt TDDA-DNNS. The membranes wereprepared by solvent casting, using THF as solvent. The membranes werecut with a cork borer (6-mm diameter) from the parent membrane andincorporated into a Philips electrode body (IS-561). The inner fillingsolution consisted of 0.1 M NaCl in 10 mM Tris-HCl buffer (pH=7.4) andwas contacted with an internal Ag/AgCl electrode. The electrodes wereconditioned overnight before experiments in the solution identical toinner filling solution. A double-junction Ag/AgCl electrode with a 1 MLiOAc bridge electrolyte was used as an external reference electrode.

Pulstrode measurements were conducted in a three-electrode cell systemwhere the Philips body electrode (acted as a working electrode),external reference electrode and counter electrode (platinum wire) wereimmersed into the sample. The pulsed galvanostatic/potentiostatictechnique was utilized in the control of the ion-selective membrane.During the experiments, each applied constant current pulse with currentdensity of 0.5 μA/cm² (of 0.5-s duration) was followed by anotherconstant zero current pulse (of 0.5-s duration), then a constantpotential pulse (of 15-s duration) was added. Sampled potentials, whichrepresent the sensor response, were obtained as the average value duringthe last 50 ms of the first current pulse.

Protamine has high content of basic amino acids, in which about 50% havearginine residues. Therefore, it is a good substrate for trypsindigestion reaction. As presented in FIG. 12 a and FIG. 12 b, theresponse of protamine decreased dramatically, upon addition of highconcentration of protease trypsin, which is indicative of the occurrenceof proteolytic reaction. All the experiments were performed using thesame electrode, and the membrane is washed with the same buffer asbackground solution between measurements with different concentrationsof trypsin. The total shift of baseline is about 7 mV, indicating theeffective refreshment of sensing membrane.

The reaction rate of trypsin digestion of protamine can be estimatedfrom the slope of the initial potential decrease upon trypsin addition.A gradually decreasing slope implied the rising reaction rate uponincreased concentration of trypsin. Therefore, the protamine digestionis shown to be directly proportional to the trypsin activity in thesample.

The activity of protease inhibitor is detected directly usingpotentiometric protamine sensor before and the trypsin-like inhibitoraprotinin was measured successfully in pretreated plasma samples. Theactivity of trypsin soybean inhibitor is estimated with Pulstrodeprotamine sensor. The inhibited reaction rate was assessed by thepotential and protamine concentration change in the course of the first96 seconds after addition the mixture of trypsin and soybean inhibitorwith a fixed trypsin concentration of 50 units per milliliter. Theresults are illustrated versus the concentration of soybean inhibitor inFIGS. 13 a and 13 b. FIG. 13 a shows the potential change versus theconcentration of the soybean inhibitor, and FIG. 13 b shows theprotamine concentration change versus the concentration of the soybeaninhibitor. The reaction rate was shown to decrease upon increasedconcentration of soybean inhibitor.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing description. Therefore, it is to be understood that theinventions are not to be limited to the specific embodiments disclosedand that modifications and other embodiments are intended to be includedwithin the scope of the appended claims.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

1. A sensor comprising: an electrode positioned within a housing; and amembrane disposed at one end of the housing and in contact with a samplesolution external to the housing, wherein the membrane detects a polyionconcentration within the sample solution, and wherein a rate ofdecomposition of the polyion concentration is directly proportional toan enzyme activity within the sample solution.
 2. The sensor accordingto claim 1, wherein the electrode is a Ag/AgCl electrode.
 3. The sensoraccording to claim 1, wherein the membrane has a surface area of about10 mm² to about 100 mm².
 4. The sensor according to claim 3, wherein themembrane has a surface area of about 20 MM² to about 50 mm².
 5. Thesensor according to claim 1, wherein the membrane has an averagethickness of about 10 μm to about 1000 μm.
 6. The sensor according toclaim 5, wherein the membrane has an average thickness of about 20 μm toabout 300 μm.
 7. The sensor according to claim 1, wherein the polyionconcentration is that of protamine.
 8. The sensor according to claim 1,wherein the enzyme activity is that of trypsin.
 9. A method of detectingpolyion concentration in a sample solution comprising: providing asample solution; and contacting the sample solution with a membrane,wherein the membrane detects a polyion concentration within the samplesolution, and wherein a rate of decomposition of the polyionconcentration is directly proportional to an enzyme activity within thesample solution.
 10. The method according to claim 9, wherein thepolyion concentration is that of protamine.
 11. The method according toclaim 9, wherein the enzyme activity is that of trypsin.
 12. The methodaccording to claim 9, wherein the sample solution comprises a biologicalcomponent.
 13. The method according to claim 9, wherein the samplesolution is blood.
 14. A reversible electrochemical cell apparatuscomprising: a polyion-selective membrane electrode comprising amembrane, wherein the membrane detects a polyion concentration withinthe sample solution, and wherein a rate of decomposition of the polyionconcentration is directly proportional to an enzyme activity within thesample solution; and means for applying a potential to clear themembrane.
 15. The reversible electrochemical cell apparatus of claim 14,wherein the polyion concentration is that of protamine.
 16. Thereversible electrochemical cell apparatus of claim 14, wherein theenzyme activity is that of trypsin.
 17. The reversible electrochemicalcell apparatus of claim 14, wherein the means for applying a potentialto clear the membrane is an external electrode potential applied betweena reference electrode and the polyion-selective membrane electrode. 18.A reversible electrochemical cell apparatus comprising: apolyion-selective membrane electrode comprising a membrane, wherein themembrane detects a polyion concentration within the sample solution; andmeans for applying a potential between a reference electrode and thepolyion-selective membrane electrode to clear the membrane.
 19. Areversible electrochemical cell apparatus comprising: apolyion-selective membrane electrode comprising a membrane, wherein themembrane detects a polyion concentration within the sample solution, andwherein a rate of decomposition of the polyion concentration is directlyproportional to an enzyme activity within the sample solution; and meansfor applying a potential between a reference electrode and thepolyion-selective membrane electrode to clear the membrane.
 20. Thereversible electrochemical cell apparatus of claim 19, wherein thepolyion concentration is that of protamine.
 21. The reversibleelectrochemical cell apparatus of claim 19, wherein the enzyme activityis that of trypsin.
 22. A method of reversing an electrochemical cellapparatus comprising: providing a sample solution; contacting the samplesolution with a polyion-selective membrane electrode comprising amembrane, wherein the membrane detects a polyion concentration withinthe sample solution, and wherein a rate of decomposition of the polyionconcentration is directly proportional to an enzyme activity within thesample solution; and applying a potential to clear the membrane.
 23. Themethod according to claim 22, wherein the polyion concentration is thatof protamine.
 24. The method according to claim 22 wherein the enzymeactivity is that of trypsin.
 25. The method according to claim 22,wherein the sample solution comprises a biological component.
 26. Themethod according to claim 22, wherein the sample solution is blood. 27.The method according to claim 22, wherein the potential for clearing themembrane is applied between a reference electrode and thepolyion-selective membrane electrode.
 28. A sensor comprising: anelectrode positioned within a housing; and a membrane disposed at oneend of the housing and in contact with a sample solution external to thehousing, wherein the membrane monitors an enzyme activity and acorresponding enzyme inhibitor activity in the sample solution.
 29. Thesensor according to claim 28, wherein the electrode is a Ag/AgClelectrode.
 30. The sensor according to claim 28, wherein the membranehas a surface area of about 10 mm² to about 100 mm².
 31. The sensoraccording to claim 30, wherein the membrane has a surface area of about20 mm² to about 50 mm².
 32. The sensor according to claim 28, whereinthe membrane has an average thickness of about 10 μm to about 1000 μm.33. The sensor according to claim 32, wherein the membrane has anaverage thickness of about 20 μm to about 300 μm.
 34. The sensoraccording to claim 28, wherein the enzyme activity is that of trypsin.35. The sensor according to claim 28, wherein the corresponding enzymeinhibitor activity is that of α1-antiproteinase inhibitor.
 36. Thesensor according to claim 28, wherein the corresponding enzyme inhibitoractivity is that of α2-macroglobulin.
 37. The sensor according to claim28, wherein the corresponding enzyme inhibitor activity is that ofaprotinin.
 38. The sensor according to claim 28, wherein thecorresponding enzyme inhibitor activity is that of a soybean inhibitor.39. The sensor according to claim 28, wherein a potential decrease isdependent on the concentration of the corresponding enzyme inhibitor inthe sample solution.
 40. The sensor according to claim 28, furtherincluding an immunoassay for detecting analytes labeled by a marker. 41.The sensor according to claim 40, wherein the marker is a polyion. 42.The sensor according to claim 40, wherein the marker is an enzyme.